Among alternative splicing events in the human transcriptome, tandem NAGNAG acceptor splice sites represent an appreciable proportion. Both proximal and distal NAG can be used to produce two splicing isoforms differing by three nucleotides. In some cases, the upstream exon can be alternatively spliced as well, which further increases the number of possible transcripts. In this study, we showed that NAG choice in tandem splice site depends considerably not only on the concerned acceptor, but also on the upstream donor splice site sequence. Using an extensive set of experiments with systematically modified two-exonic minigene systems of AFAP1L2 or CSTD gene, we recognized the third and fifth intronic upstream donor splice site position and the tandem acceptor splice site region spanning from -10 to +2, including NAGNAG itself, as the main drivers. In addition, competition between different branch points and their composition were also shown to play a significant role in NAG choice. All these nucleotide effects appeared almost additive, which explained the high variability in proximal versus distal NAG usage.

• MeSH
• alternativní sestřih genetika   MeSH
• exony genetika   MeSH
• HeLa buňky MeSH
• introny genetika   MeSH
• lidé MeSH
• místa sestřihu RNA genetika   MeSH
• nádorové buněčné linie MeSH
• nukleotidy genetika   MeSH
• tandemové repetitivní sekvence genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Pre-mRNA splicing is an essential step in gene expression, when introns are removed and exons joined by the complex of proteins called spliceosome. Correct splicing requires a precise exon/intron junction definition, which is determined by a consensual donor and acceptor splice site at the 5' and 3' end, respectively. An acceptor splice site (3'ss) consists of highly conserved AG nucleotides in positions E-2 and E-1. These nucleotides can appear in tandem, located 3 bp from each other. Then they are referred to as NAGNAG or tandem 3'ss, which can be alternatively spliced. NAG/TAG 3'ss motif abundance is extremely low and cannot be easily explained by just a nucleotide preference in this position. We tested artificial NAG/TAG motif's potential negative effect on exon recognition using a minigene assay. Introducing the NAG/TAG motif into seven different exons revealed no general negative effect on exon recognition. The only observed effect was the partial use of the newly formed distal 3'ss. We can conclude that this motif's extremely low preference in a natural 3'ss is not a consequence of the NAG/TAG motif's negative effect on exon recognition, but more likely the result of other RNA processing aspects, such as an alternative 3'ss choice, decreased 3'ss strength, or incorporating an amber stop codon.

• MeSH
• alternativní sestřih MeSH
• exony * MeSH
• HeLa buňky MeSH
• introny MeSH
• lidé MeSH
• místa sestřihu RNA genetika   fyziologie   MeSH
• nukleotidy genetika   MeSH
• sekvence nukleotidů MeSH
• sestřih RNA genetika   MeSH
• tandemové repetitivní sekvence MeSH
• terminační kodon MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.

• MeSH
• alternativní sestřih * MeSH
• exony genetika   MeSH
• genetická variace MeSH
• geny BRCA1 * MeSH
• geny BRCA2 * MeSH
• komplementární DNA MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• místa sestřihu RNA * MeSH
• mutace MeSH
• nádory prsu genetika   MeSH
• nádory vaječníků genetika   MeSH
• počítačová simulace MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought.

• MeSH
• exony * MeSH
• HeLa buňky MeSH
• lidé MeSH
• místa sestřihu RNA * MeSH
• RNA malá jaderná * metabolismus   genetika   MeSH
• sekvence nukleotidů MeSH
• sestřih RNA MeSH
• transkripční faktor STAT3 * metabolismus   genetika   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Prosaposin deficiency (pSap-d) and saposin B deficiency (SapB-d) are both lipid storage disorders caused by mutations in the PSAP gene that codes for the 65-70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A-D. We report on two new patients with PSAP gene defects; one, with pSap-d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB-d, who presented with a metachromatic leukodystrophy-like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap-d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap-d patient was homozygous for a splice-acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB-d patient was a compound heterozygote with a splice-acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap-d mutation, on the other allele. Phenotypically, pSap-d is a relatively uniform disease of the neonate, whereas SapB-d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap-d and the single saposin deficiencies is speculated.

• MeSH
• dítě MeSH
• financování organizované MeSH
• heterozygot MeSH
• homozygot MeSH
• kojenec MeSH
• kůže patologie   MeSH
• lidé MeSH
• magnetická rezonanční tomografie MeSH
• metachromatická leukodystrofie genetika   metabolismus   patologie   MeSH
• místa sestřihu RNA genetika   MeSH
• mozek abnormality   patologie   MeSH
• mutace MeSH
• mutační analýza DNA MeSH
• nesmyslný kodon MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• saposiny genetika   nedostatek   MeSH
• sekvenční delece MeSH
• sfingolipidy moč   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• Publikační typ
• kazuistiky MeSH

• MeSH
• bodová mutace MeSH
• dyneiny genetika   MeSH
• inzerční mutageneze MeSH
• lidé MeSH
• missense mutace MeSH
• místa sestřihu RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• myosin VIIa MeSH
• myosiny genetika   MeSH
• nesmyslný kodon MeSH
• polymorfismus konformace jednovláknové DNA MeSH
• rodokmen MeSH
• sekvence aminokyselin MeSH
• sekvenční delece MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• tandemové repetitivní sekvence MeSH
• Usherovy syndromy etnologie   genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Geografické názvy
• Česká republika MeSH
• Itálie MeSH
• Maroko MeSH
• Španělsko MeSH
• Turecko MeSH

• MeSH
• DNA genetika   MeSH
• genetické jevy MeSH
• genom MeSH
• geny fyziologie   MeSH
• proteiny genetika   MeSH
• RNA genetika   MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Obecná genetika. Obecná cytogenetika. Evoluce
• NLK Obory
• genetika, lékařská genetika